doi: 10.1038/nature07182 SUPPLEMENTAL FIGURES AND TABLES Fig. S1. myf5-expressing cells give rise to brown fat depots and skeletal muscle (a) Perirenal BAT from control (cre negative) and myf5-cre:r26r3-yfp mice were analyzed by immunohistochemistry to detect YFP (anti-gfp) and UCP1 protein. (b) Real-time PCR analysis of YFP gene activation in: heart, lung, spleen, kidney, interscapular BAT (IBAT), axial BAT (ABAT) and gastrocnemius muscle (Gast.) from myf5-cre:r26r3-yfp mice (c) Real time PCR analysis of PRDM16, UCP1, myf5 and cre recombinase expression in WAT, BAT and skeletal muscle (skm) (n=4-6 mice/group; error bars represent ± SEM). **p<0.01. www.nature.com/nature 1
Fig. S2. PRDM16 expression blocks myogenic differentiation (a) C2C12 myoblast cultures expressing retroviral PRDM16 or vector control (ctl) were induced to undergo muscle differentiation in 2% horse serum and analyzed by immunocytochemistry for expression of skeletal Myosin Heavy Chain (MyHC) protein. (b) Real-time PCR analysis of skeletal muscle-specific genes (n=3; error bars represent± SD; *p<0.05 **p<0.01). Fig. S3. PRDM16 coactivates exogenous PPARγ in PPARγ-deficient cells Transcriptional activity of a PPAR-driven reporter gene in response to PRDM16 or vector expression in PPARγ-/- cells with or without exogenous PPARγ/RXRα (+/- 1 μm rosiglitazone) (n=3; error bars represent ± SD; **p<0.05). www.nature.com/nature 2
Fig. S4. PRDM16 binds and activates the transcriptional function of PPARα (a) Flag-PPARα was immunoprecipitated from COS-7 cells expressing exogenous PRDM16 and/or Flag-PPARα followed by western blot analysis to detect PRDM16. The interaction was examined in the presence or absence of 1 μm of a specific PPARα ligand, WY-14643 (Wy). (b) Transcriptional activity of a PPAR-driven reporter gene (3x DR1) in response to PPARα/RXRα and PRDM16 or vector expression in COS-7 cells (+/- 1 μm Wy). (n=3; error bars represent ± SD; **p<0.05). Fig. S5. PPARγ activation is required for the adipogenic action of PRDM16 (a) Oil-Red-O staining of C2C12 myoblast cultures expressing retroviral PRDM16 or vector control (ctl) 6 days after inducing adipocyte differentiation in the presence or absence of 1 μm of the PPARγ-specific agonist, rosiglitazone. (b) Real-time PCR analysis of: PRDM16; adipocyte markers (PPARγ, ap2, Adiponectin); and BAT-selective genes (CIDEA, UCP1) (n=3; error bars represent ± SD; **p<0.05). www.nature.com/nature 3
doi: 10.1038/nature07182 Fig. S6. PGC-1β expression does not stimulate adipogenesis in myoblasts (a) Oil-Red-O staining of C2C12 myoblast cultures transduced with retroviral PRDM16, PGC-1β or vector control 7 days after inducing adipocyte differentiation. (b) Real-time PCR analysis of: adipocyte markers (left), BAT genes (UCP1, CIDEA); and mitochondrial genes (cyc, cox5b, cox4i1) (n=3, error bars are ± SD; *p<0.05 **p<0.01). Fig. S7. PPARγ alone drives adipogenesis but does not induce the brown fat cell gene program in C2C12 myoblasts (a) Oil-Red-O staining of C2C12 myoblast cultures expressing retroviral PRDM16, PPARγ2 or empty vector control (ctl) 7 days after induction of adipocyte differentiation. (b) Real-time PCR analysis of: adipocyte markers; BATselective genes; and skeletal muscle-specific genes (n=3 per group, error bars represent ± SD; *p<0.05 **p<0.01). www.nature.com/nature 4
Fig. S8. Model of PRDM16 function in the specification of brown fat fate Lineage tracing studies reveal that myf5-expressing precursors give rise to skeletal muscle and BAT but not WAT or any other tissues examined. PRDM16 expression in myoblasts induces BAT development and represses skeletal muscle differentiation. PRDM16 stimulates brown adipogenesis in myoblasts, at least in part, via binding and coactivating PPARγ. Together, these studies suggest that PRDM16 expression in bipotent muscle/brown fat progenitor cells determines brown fat identity. The mechanisms by which PRDM16 promotes brown fat determination and represses the muscle lineage in this upstream progenitor cell remain to be defined. www.nature.com/nature 5
Table S1. MW Proteins identified by MALDI-TOF-MS/MS Protein Identification ~30 kda splicing factor, arginine/serine-rich 1 isoform 2 (~22 KDa); NCBI# 26383576; Sequence: I Y V G N L P P D I R ~35 kda ribosomal protein L6 (~33 kda); NCBI# 84662736; Sequence: A V P Q L Q G Y L R ~42 kda A: β-actin (~42 kda); NCBI# 74190672 B: tropomodulin 3 (~40 kda); NCBI# 8394460 ; Sequence: F G Y Q F T Q Q G P R ~48 kda A: β -actin (~42 kda); NCBI# 74190672 B: C-terminal binding protein 1 (~48 kda); NCBI# 7304989 C: C-terminal binding protein 2 (~46 kda); NCBI# 2909779 Sequence: I R G E T L G L I G F G R; D: heterogeneous nuclear ribonucleoprotein F (~46 kda); NCBI# 19527048 Sequence: H S G P N S A D S A N D G F V R ~55 kda A: Trim35 (~55kda); NCBI # 32425788; Sequence: I R D E F D K L R B: polymerase I and transcript release factor (~43kda); NCBI # 6679567 Sequence: K S F T P D H V VYAR C: heterogeneous nuclear ribonucleoprotein H2 (~49 kda); NCBI# 9845253 Sequence: H T G P N S P D T A N D G F V R D: immunoglobulin heavy chain constant region (~36 kda); NCBI# 14091948 Sequence: A P Q V Y T I P P P K E Q M A K ~65 kda A: DEAD box polypeptide 5 (~69 kda); NCBI# 120538559 B: PPARγ (~57kDa); NCBI # 18255316 Sequence: H I T P L Q E Q S K ~70 kda A: HSP 70, protein 5 (~72 kda); NCBI# 31981722 B: HSP 8 (~71kDa); NCBI# 42542422 C: HSP 9A (~74 kda); NCBI# 6754256 D: heterogeneous nuclear ribonucleoprotein M (~78 kda); NCBI# 21313308 ~85 kda gelsolin (~86kDa); NCBI# 28916693 ~100 kda A: myosin IC isoform b (~118kDa); NCBI# 6678986 B: major vault protein (MVP); NCBI # 17433104 Sequence: V P H N A A V Q V Y D Y R; C: gelsolin (~86kDa); NCBI# 28916693 ~125 kda pyruvate carboxylase (~130 kda); NCBI# 6679237 ~150 kda PRDM16 protein (~130 kda); NCBI# 37590584 ~175 kda PRDM16 protein (~130 kda); NCBI# 37590584 ~200 kda myosin, heavy polypeptide 9, non-muscle (~226 kda); NCBI# 114326446 ~300 kda A: α-spectrin (~270kDa); NCBI # 115496850 B: β-spectrin (~270kDa); NCBI # 117938332 Note: All samples contained peptides derived from PRDM16; this information was removed before identifications were made for each protein sample. www.nature.com/nature 6
Table S2. Primers used for real-time PCR analysis Gene Forward primer (5-3 ) Reverse primer (5-3 ) adiponectin GCACTGGCAAGTTCTACTGCAA GTAGGTGAAGAGAACGGCCTTGT ap2 ACA CCG AGA TTT CCT TCA AAC TG CCA TCT AGG GTT ATG ATG CTC TTC A cidea TGC TCT TCT GTA TCG CCC AGT GCC GTG TTA AGG AAT CTG CTG cox4i1 ACCAAGCGAATGCTGGACAT GGCGGAGAAGCCCTGAA cox5b GCTGCATCTGTGAAGAGGACAAC CAGCTTGTAATGGGTTCCACAGT cox7a1 CAG CGT CAT GGT CAG TCT GT AGA AAA CCG TGT GGC AGA GA cox8b GAA CCA TGA AGC CAA CGA CT GCG AAG TTC ACA GTG GTT CC Cre GCG GTC TGG CAG TAA AAA CTA TC GTG AAA CAG CAT TGC TGT CAC TT cyc GCAAGCATAAGACTGGACCAAA TTGTTGGCATCTGTGTAAGAGAATC elovl3 TCC GCG TTC TCA TGT AGG TCT GGA CCT GAT GCA ACC CTA TGA glut4 GTG ACT GGA ACA CTG GTC CTA CCA GCC AGT TGC ATT GTA G MCK GCA AGC ACC CCA AGT TTG A ACC TGT GCC GCG CTT CT MHC TCC AAA CCG TCT CTG CAC TGT T AGC GTA CAA AGT GTG GGT GTG T (embryonic) myod CGC CAC TCC GGG ACA TAG GAA GTC GTC TGC TGT CTC AAA GG myg AGC GCA GGC TCA AGA AAG TGA ATG CTG TAG GCG CTC AAT GTA CTG GAT myf5 CAG CCC CAC CTC CAA CTG GGG ACC AGA CAG GGC TGT TA myf6 ATC AGC TAC ATT GAG CGT CTA CA CCT GGA ATG ATC CGA AAC ACT TG otopetrin1 ACT AGG ACC CCG TCG AAT CT ACC ATG CTC TAC GTG CTG TG PGC-1α CCC TGC CAT TGT TAA GAC C TGC TGC TGT TCC TGT TTT C PGC-1β TCC TGT AAA AGC CCG GAG TAT GCT CTG GTA GGG GCA GTG A PPARγ GTGCCAGTTTCGATCCGTAGA GGCCAGCATCGTGTAGATGA PRDM16 CAG CAC GGT GAA GCC ATT C GCG TGC ATC CGC TTG TG TBP GAA GCT GCG GTA CAA TTC CAG CCC CTT GTA CCC TTC ACC AAT UCP1 ACT GCC ACA CCT CCA GTC ATT CTT TGC CTC ACT CAG GAT TGG YFP CACGACTTCTTCAAGTCCGCCATG GCGGATCTTGAAGTTCACCTTGAT www.nature.com/nature 7